Biosafety Considerations for Autopsy

Kurt B. Nolte, M.D., David G. Taylor, Ph.D., and Jonathan Y. Richmond, Ph.D.

An autopsy may subject prosectors and others to a wide variety “. . .What kind of a cut is it? Where is it?” of infectious agents, including bloodborne and aerosolized “Right here, on my finger. I rode over to the village pathogens such as human immunodeficiency virus, hepatitis B today—you know, the one they brought that and C viruses, and Mycobacterium tuberculosis. Other hazards include toxic chemicals (e.g., formalin, cyanide, and organo- mouzhik with typhus from. For some reason or phosphates) and radiation from radionuclides used for patient other they were getting ready to do an autopsy on therapy and diagnosis. These risks can be substantially miti- him, and it’s been a long time since I’ve had any gated through proper assessment, personal protective equip- practice of that sort.” ment, appropriate autopsy procedures, and facility design. “. . .Old timer,” Bazarov began in a hoarse, slow Key Words: Autopsy—Biohazards—Chemical hazards— Toxichazards. voice, “my goose is cooked; I’ve been infected and in a few days you’ll be burying me.” Ivan S. Tur- genev, Fathers and Sons, 1862 Autopsies can be performed with the consent of the next-of-kin of persons who die of natural causes in hospitals, or they can be performed under legislated authority (forensic or medicolegal autopsies) on persons who die of violent, unnatural, suspicious, sudden, or unexplained causes. The frequency of consent autopsies has declined substantially over the previous several de- cades, from approximately 50% of all hospital deaths in 1950 to less than 10% in 1995 (1). One reason for this decline is the potential increased risk of occupational exposure of pathologists to dangerous pathogens (2). With decreased hospital autopsy rates, the proportion of medicolegal postmortem examinations has increased. Medicolegal autopsies constituted a large proportion, and in some jurisdictions the majority, of the total number performed in 1981 (3). Since then, national hospital autopsy rates have continued to decline, and medicolegal autopsies likely represent the majority of these procedures performed in almost all areas of the United States.

AUTOPSY HAZARDS Manuscript received January 20, 2002; accepted January 20, 2002. From the Office of the Medical Investigator, University of New Mexico School of Medicine, Albuquerque, New Mexico, the Medical Examiner/Coroner Information Sharing Program, Division of Public Infectious Agents Health Surveillance and Informatics, Epidemiology Program Office, The risk of infectious disease transmission has long and Infectious Disease Pathology Activity, Division of Viral and Rick- been recognized for prosectors, observers, and other per- ettsial Diseases, National Center for Infectious Diseases (K.B.N.) and the Office of Health and Safety (D.G.T., J.Y.R.), Centers for Disease sons in close proximity to an autopsy. Retrospective Control and Prevention, Atlanta, Georgia, U.S.A. surveys of British clinical laboratories between 1970 and Address correspondence and reprint requests to Kurt B. Nolte, M.D., 1989 demonstrated that the highest rates of laboratory- Office of the Medical Investigator, University of New Mexico School of Medicine, Albuquerque, NM 87131-5091, U.S.A.; e-mail: knolte@ acquired infections were in autopsy workers (4–8). Au- salud.unm.edu topsy-transmitted infections may occur after direct cuta-

107 108 K. NOLTE ET AL. neous inoculation (percutaneous injury), contact with per exposure (31); and for HCV, an average of 1.8% per droplets, and aerosol exposure. exposure but may be as high as 10% (38,46). Although Pathology residents sustained a percutaneous injury the viability of HIV in cadaveric blood appears to de- with blood exposure in 1 of 11 autopsies and experienced crease over time, this organism has been isolated from pathologists in 1 of 55 autopsies (9). Autopsy prosectors specimens from deceased persons with postmortem in- sustained cuts at twice the frequency that they sustained tervals of 6, 11, and 16 days (47–50). HIV-infected needle punctures (9). Scalpel blades created the majority bodies should be considered infectious for at least 2 of these cuts, resulting in a potentially large inoculum of weeks after death. HBV in the environment is also hardy. infectious agent. However, many other sharp objects HBV in human plasma retained infectivity 1 week after such as broken glass, embedded needle fragments, bone being dried and exposed to an ambient environment (51). shards, and fragmented projectiles can injure autopsy Clearly, the transmission risks for these bloodborne personnel (9–11). In addition, approximately 8% of sur- pathogens, combined with their high seroprevalence in gical gloves are punctured during autopsy, and approxi- certain autopsy populations and the frequency of percu- mately one third of these punctures remain undetected by taneous injury, place autopsy personnel at high risk for the prosector (12). Glove punctures may cause preexist- sustaining an occupational infection. ing hand lesions to be bathed in infectious blood for Performing autopsies on persons who have died of prolonged periods of time. viral hemorrhagic fever (VHF) poses even greater risks. Many infections can be transmitted by direct inocula- Prosectors have died of autopsy-transmitted Marburg, tion. For example, pathologists have died of streptococ- Ebola, and Lassa hemorrhagic fevers (22,26,52,53). cal sepsis after sustaining minor cutaneous injuries dur- These infections have been transmitted by direct cutane- ing autopsies on persons with the same disease (13). ous inoculation. Although aerosol transmission of VHF Other infections that can be transmitted in this manner has been suspected in outbreaks occurring within hospi- include tuberculosis, blastomycosis, coccidioidomycosis, tals (54,55), whether these infections may also be trans- acquired immunodeficiency syndrome, hepatitis B and C mitted via autopsy aerosols is unclear. Lymphocytic cho- (or non-A, non-B), rabies, tularemia, diphtheria, erysip- riomeningitis and yellow fever have been fatally eloid fever, and some of the viral hemorrhagic fevers transmitted to human prosectors, and Rift Valley fever (4,8,14–33). Some of these autopsy-transmitted infec- has been transmitted to prosectors of veterinary case tions have proved to be fatal. material (56–58). None of these persons were reported to Among physicians, pathologists are recognized as a have sustained an injury during dissection. The prosec- high-risk group for occupationally acquired hepatitis B tors in whom Rift Valley fever developed did not wear virus (HBV) because of their exposure to blood (34,35). masks during the procedure. Autopsy aerosols could There are at least two records of autopsy workers who have transmitted these infections. The conditions where died of occupationally acquired hepatitis (8,32). The autopsies are performed are often primitive in most lo- prevalence of HBV, hepatitis C virus (HCV), and human cations where deaths resulting from VHF occur. There- immunodeficiency virus (HIV) infection is higher in fore, the risks and benefits must be carefully considered forensic autopsy populations than in the general public before an autopsy is performed on a person suspected to because of an overrepresentation of intravenous drug have died of one of these conditions (19). Immunohis- abusers among decedents subjected to autopsy (36,37). tochemical procedures on formalin-fixed skin biopsy In some areas of the United States, up to 90% of intra- specimens have proved useful in the diagnosis of Ebola venous drug abusers may be infected with HCV (38). In hemorrhagic fever (52). A skin biopsy can be performed one study of the autopsy population in Baltimore, Mary- more safely and readily than an autopsy, thereby reduc- land, the medical examiner demonstrated an infection ing exposure to infectious materials. However, if the skin seroprevalence of 5.6% for HIV, 23.2% for HBV, and biopsy result is negative and no autopsy has been per- 19.1% for HCV (37). This study did not discriminate formed, definitive diagnosis may be impossible. Autop- between acute and remote HBV infections. Another sies have been performed safely on deceased persons study of a medicolegal cadaver population in Milan, with VHF, using strict safety protocols (59). Similar to Italy, revealed an infection seroprevalence of 16% for the diagnosis of VHF, immunofluorescence and immu- HIV and 29% for HCV (39). Other studies of forensic nohistochemical procedures can be used to detect rabies autopsy populations have identified an HIV seropreva- virus antigen in skin and may obviate the risks associated lence of 2% in Vancouver, Canada (40), 11% in South with an autopsy (60,61). Africa (41), 2.2% in Philadelphia, Pennsylvania (42), Autopsies on persons who died of hantavirus infec- and 1% in Scotland (43). In 1983, 18% of young adults tions appear to pose fewer risks than other VHFs. A who died suddenly in San Francisco had antibodies to study of health care workers (including autopsy prosec- HIV (44). The transmission risk with infected blood for tors) involved in a 1993 hantavirus pulmonary syndrome HIV is 0.3% per exposure (45); for HBV, at least 30% outbreak indicated no evidence of autopsy transmission

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(62). Nonetheless, strict adherence to Universal Precau- portion of medical students became tuberculin skin test– tions and the additional use of N-95 respirators are rec- positive, and fatal tuberculosis developed in several of ommended (63). them, after the autopsy training period of their curricu- Spongiform encephalopathies such as Creutzfeldt-Ja- lum (84,85). kob disease (CJD) also can be transmitted by percutane- An autopsy is an exceptionally efficient method of ous autopsy exposure. These transmissible dementias are transmitting tuberculosis from the decedent to those caused by infectious isoforms of host membrane sialo- present in the dissection room. Eight of 35 Mantoux- glycoproteins known as prion proteins (64). No clearly negative medical students exposed to a tuberculosis au- documented cases of occupational CJD among general topsy for 1 hour became infected. The risk for infection pathologists, neuropathologists, laboratory and autopsy did not vary with distance from the autopsy table (86). technicians, or morticians exist (65); however, two cases Autopsy exposures as brief as 10 minutes have resulted of CJD have been documented in histology technicians in transmission of tuberculosis to an observing medical working in separate neuropathology laboratories (66,67). student (87). Unprotected autopsy prosectors exposed to Whether these cases represent an occupational exposure unsuspected tuberculosis often have a higher risk for or another unknown exposure is unclear. contracting this infection than clinicians who cared for Although no evidence exists of contact or aerosol the patient before death (87,88). transmission, the absence of any known effective treat- Periodic retrospective surveillance of British clinical ment of prion diseases demands caution during the han- laboratories between 1970 and 1989 revealed six cases of dling of infected tissues. Recommendations for safely occupationally acquired tuberculosis in autopsy workers conducting autopsies, handling and processing tissues, (4). Between 1953 and 1955, British autopsy patholo- and decontaminating instruments and work surfaces are gists had the highest claim rates for disability benefits given at the end of this article. Note that prions are not resulting from occupational tuberculosis. For this same inactivated by formalin (a routine fixative used in pre- group, between 1949 and 1953, the incidence of dis- paring samples for pathologic examination) and retain abling tuberculosis ranged from three times (men) to transmissibility in paraffin blocks (68). eight times (women) the incidence in the general popu- Infectious aerosols are composed of airborne particles lation. The incidence remained high when investigators approximately 1 to 5 ␮m in diameter, which can remain controlled for socioeconomic status (89). Teppo et al. suspended in air for long periods of time. When inhaled, (90), in a survey of Finnish pathologists spanning a wide the particles traverse the upper respiratory passages and range of practice years, reported that active tuberculosis reach the pulmonary alveoli (69). Particles with diame- attributable to autopsy performance developed in 10%. ters larger than 5 ␮m (i.e., droplets) can also pose a risk The occupational tuberculosis rate of pathologists was for autopsy participants. However, droplets have less substantially higher than that of clinicians (1%) and potential for traveling substantial distances beyond the specialists in tuberculosis and pulmonology (4%). Sim- autopsy area. Aerosols are generated by fluid aspirator ilarly, in Japan, pathologists and pathology technicians hoses vented into sinks, oscillating saws applied to bones who engaged in autopsies were 6 to 11 times more likely and soft tissues, and water sprayed by hoses onto tissue to experience occupational tuberculosis than were non- surfaces (70,71). Even compressing and dissecting lungs autopsy workers in pathology departments and workers with standard autopsy tools can create infectious aerosols in university departments of preventive medicine and and droplets (72). Oscillating saws produce large quan- public health (91). tities of respirable dust and bacteria when they are ap- It is not unusual for tuberculosis to remain undetected plied to bone (71,73). Concentrations of respirable bone until a patient dies. Of all tuberculosis cases reported in dust up to 5700 particles/ml have been measured in the the United States between 1985 and 1988, 5.1% (4,541 breathing zone of autopsy workers using oscillating saws cases) were recognized post mortem (92). In a study of (71). Infectious HIV has been recovered from aerosols hospital autopsies from New York City, 4% of cases in created experimentally by applying oscillating saws to which tuberculosis was the cause of death were undiag- infected blood (74). Given the uniform use of oscillating nosed before autopsy (93). However, 50% of autopsied saws and spray and aspirator hoses by prosectors, a fair active tuberculosis cases in hospitals in Dundee, Scot- assumption is that all autopsies generate potentially in- land, were unrecognized before autopsy (94). Because fectious aerosols. autopsy cases in hospitals usually have more clinical Mycobacterium tuberculosis is the prototypical organ- diagnostic information available before the autopsy than ism transmitted by autopsy-generated aerosols. How- most medicolegal autopsies, the percentage of unrecog- ever, these aerosols can also potentially transmit other nized tuberculosis cases in a forensic autopsy population infections, including rabies, plague, legionellosis, menin- would likely be substantially higher. gococcemia, rickettsioses (e.g., Q fever), coccidioidomy- In recent years, outbreaks of autopsy-transmitted tu- cosis, and anthrax (21,75–83). Historically, a large pro- berculosis have occurred in the Syracuse Medical Exam-

Am J Forensic Med Pathol, Vol. 23, No. 2, April 2002 110 K. NOLTE ET AL. iner’s Office, the Los Angeles Coroner’s Office, the breathing a concentration that exceeds the occupational University of Arkansas School of Medicine, and the standard. Long-term inhalation of this substance has University of Health Sciences/Chicago Medical School been associated with an increased risk for all cancers and (87,88,95,96). Multidrug-resistant M. tuberculosis cancer of the lung as a function of cumulative exposure caused the Syracuse outbreak, which was attributed to a (108). An association between chronic formaldehyde ex- positive pressure autopsy room, where exhausted room posure and cancer of the nasal passages remains contro- air circulated throughout the facility (95). Similarly, the versial (105,109). Interestingly, mortality studies of pa- outbreak in the Los Angeles Coroner’s Office was attrib- thologists and laboratory technicians demonstrated no uted to inadequate ventilation (96). However, tuberculo- carcinomas of the nasal passages and a decreased risk of sis can be efficiently transmitted to autopsy prosectors in lung cancer compared with the general population facilities that have adequate ventilation when proper (32,110). However, these studies did not evaluate their personal respiratory protection is not used (88). Cultures smoking history. from embalmed bodies have yielded isolates of M. tu- Forensic pathologists and their technicians are some- berculosis as long as 60 hours after fixation (97); there- times exposed to cyanide when performing autopsies on fore, the dissection of formalin-fixed tissues has the persons who have died after ingesting this substance potential to transmit infectious tuberculous aerosols. In- (111). Although cyanide can volatilize from autopsy deed, even the process of embalming has been shown to tissues, the major risk to autopsy personnel occurs when transmit tuberculous aerosols (98). the stomach is opened. In the acid gastric environment, Autopsy-generated droplets (Ͼ5 ␮m in diameter) also cyanide salts are converted to highly volatile hydrocya- have the potential to transmit infections if they are in- nic gas (112,113). Prosectors risk inhaling potentially haled or ingested. For example, glanders was fatally toxic concentrations of this gas unless the stomach is transmitted to a veterinary prosector who had a drop of opened in a totally exhausted biosafety cabinet or chem- infected horse blood enter his mouth (99). Similarly, ical fume hood (114). fatal glanders developed in a prosector as a consequence A similar inhalation danger exists with metallic phos- of smoking while he was dissecting an infected guinea phides used in commercial rodenticide pellets. In cases pig (100). of fatal metallic phosphide ingestion, phosphine (hydro- Autopsy personnel occupationally acquired other in- gen phosphide) is released in the acid medium of the fections; however, the mechanism of transmission re- stomach (115). This extremely poisonous gas causes mains unspecified. These infections have included scrub symptoms at concentrations of approximately 2 ppm typhus and toxoplasmosis (101,102). In two separate (115). Prosectors have become ill (headache and nausea) incidents, fatal smallpox developed in a prosector and an after dissecting persons with fatal metallic phosphide observer after they participated in autopsies on subjects ingestion without taking proper safety precautions (D. who had died of smallpox (103,104). The mechanism of Little, personal communication, 1999) (116). However, transmission for the smallpox cases and the precautions autopsies have also been performed safely on these cases taken by the infected persons were not specified. How- by prosectors using fume respirators and limiting their ever, the fact that smallpox was transmitted to an ob- duration of autopsy exposure (KA Margolius, personal server suggests an airborne mechanism. Fatal glanders communication, 1999). In one case of fatal metallic developed in a prosector after dissection of an infected phosphide ingestion, the autopsy risk was deemed exces- human body (99). Despite speculation about direct inoc- sive, and the procedure was contravened (PSJ Ellis, ulation and aerosolization of the organism, the mecha- personal communication, 1999). nism of transmission is unknown. Autopsy prosectors may examine persons who have died of organophosphate pesticide (e.g., malathion and Toxic Agents parathion) poisoning (116–118). These substances may The most common toxic agent to which autopsy pro- cause toxicity as a consequence of inhalation, ingestion, sectors are exposed is formaldehyde used to preserve or dermal absorption (119–122). Exposure to gastric tissues. Formaldehyde is highly volatile and causes an contents and clothing containing or contaminated with array of symptoms, including irritation of the eyes, mu- organophosphate pesticides can be dangerous (116,123). cous membranes, and skin (105,106). The Occupational The stomach should be opened in a fume hood. Prosec- Safety and Health Administration limits the occupational tors should routinely wear gloves when handling poten- exposure to this chemical to 0.75 ppm as an 8-hour tially contaminated clothing and other personal articles. time-weighted average and to 2.0 ppm for short-term Nerve gas agents, (e.g., tabun, sarin, VX, and soman) (15-minute) exposures (107). The odor threshold for are also organophosphorus compounds and are poten- formaldehyde ranges from 0.1 to 1.0 ppm and most tially weapons of chemical warfare and bioterrorism commonly is 0.5 ppm (105). Therefore, the ability to (124). These agents can slowly penetrate heavy rubber smell this substance generally means that the person is gloves and aprons and be absorbed through the skin.

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Consequently, bodies contaminated with these agents TABLE 1. Isotopes commonly used in human subjects should be thoroughly washed with water, or preferably Use Type of radiation Half-life an alkaline solution (e.g., 5.0% hypochlorite) (125). Pro- Diagnostic sectors should wear a positive pressure, pressure-de- I-131 gamma 8 days mand, full facepiece, self-contained breathing apparatus Tc-99m gamma 6 hours Ga-67 gamma 78 hours (SCBA) or pressure-demand supplied air respirator with In-111 gamma 2.8 days escape SCBA and a fully encapsulating, chemical-resis- Therapeutic tant suit with butyl/neoprene or viton/neoprene gloves I-131 gamma 8 days Sr-90 beta 28.8 years (126). Nerve gas vapors from casualties can overwhelm P-32 beta 14 days prosectors (127). Organic vapor respirators specific for Implants nerve gases offer effective protection from toxic nerve Temporary Cs-137 gamma 30 years agent or organophosphate pesticide vapors (125). Ir-192 beta 74 days A chemical used in the manufacture of certain herbi- Permanent cides is 2,4-Dichlorophenol (2,4-DCP). Chemical work- I-125 gamma 60 days Pd-103 x-ray; EC 17 days ers exposed to 2,4-DCP in its liquid state can die rapidly EC, electron capture. after dermal absorption because this substance uncouples oxidative phosphorylation (128). Prosectors handling the bodies in such fatalities must wear impermeable barrier both dimethyl sulfate (produced from dimethyl sulfoxide garments, be cautious while handling contaminated use) and chloramine (produced by mixing bleach and clothing, and thoroughly wash the decedent’s skin before urine in a hospital sink) have been hypothesized (137– the postmortem examination. 139). The autopsy in this case was safely performed in a Prosectors also may be exposed to elemental mercury. specially constructed chamber. The prosectors used air- This element is highly volatile and is well absorbed tight suits and external air supplies (137,140,141). through inhalation (129). However, no reported cases of One homicide case involved an attempt to destroy the toxicity in autopsy personnel have been documented. body by placing it in concentrated hydrochloric acid (D Phytotoxins such as abrin in the rosary pea (Abrus Little, personal communication, 1999). Such a case poses precatorius) and ricin in the castor bean (Ricinus com- the risk of both fume inhalation and cutaneous/ocular munis) are toxic in minute concentrations (130–132). splash to autopsy workers. The autopsy was safely per- Most fatalities from these substances occur from inges- formed by the use of a downdraft autopsy table; copious tion of the seeds. Seed or seed fragments within stomach washing of the body surface with water and sodium contents would likely pose no risk to autopsy prosectors. bicarbonate; and the use of face shields, water-resistant However, a homicide case thought to be a consequence polypropylene overalls with hoods, and acid-resistant of ricin poisoning involved a small partially hollowed gloves and boots. metallic sphere injected into a leg with an umbrella specifically fabricated for this purpose (133,134). Such Radiation toxin-bearing devices can pose an inoculation risk for Autopsy workers may be exposed to radioactive ma- prosectors dissecting wounds, especially if the injected terials in a body from diagnostic or therapeutic proce- devices are sharp. dures (142). A pathologist who performed an autopsy Individuals who die of botulism are generally not without precautions on an individual who had recently considered to pose a risk to autopsy personnel. Currently, undergone a 67-gallium scan received an excessive ra- aerosolized Clostridium botulinum toxin is considered to diation exposure (143). However, with proper precau- be a potential bioterrorism agent (135). Three prosectors tions, bodies containing strontium-89-chloride and sodi- experienced botulism after performing necropsies on um-131-iodide have been safely examined (144–147). guinea pigs and rabbits experimentally exposed to aero- When bodies contain isotopes that have a long half-life, solized botulinum toxin. The workers wore gloves but no such as strontium-90 (28 years), it may be preferable to other protective equipment. The toxin was thought to bury the body in a sealed casket without performing an have been aerosolized from the animals’ fur during nec- autopsy (145). ropsy (136). The application of this episode to potential The extent of radiation exposure is dependent on the human bioterrorism fatalities from aerosolized botuli- dose administered to the patient, the type of radiation num toxin is unclear. However, prosectors would be wise emitted, the radionuclide, the exposure time, and the to take aerosol precautions when handling such cases. shielding or protection used by the autopsy prosector. In 1994, a mysterious fatality in California was ini- Although the half-lives of the diagnostic radionuclides tially thought to be a consequence of a toxin that gener- are short (Table 1), their emissions are usually of the ated incapacitating fumes for emergency room person- more penetrating type (i.e., gamma rays). The therapeu- nel. The precise toxin was never identified, although tic and implant nuclides have longer half-lives and rep-

Am J Forensic Med Pathol, Vol. 23, No. 2, April 2002 112 K. NOLTE ET AL. resent a potential concern for a longer time. Exposure TABLE 2. Principles of Biosafety Level 2 time and shielding are important factors to consider for Suitable for work with agents of moderate potential hazard minimizing radiation exposure. Rubber gloves will ap- to personnel and the environment Ⅵ Personnel are trained in hazard identification and work preciably reduce beta radiation but not the more pene- procedures trating gamma radiation from isotopes (148). Ⅵ Access to work area is controlled and limited; hazard signs If a body is thought to contain radioactive materials, a are posted Ⅵ Extreme precautions with sharps are observed radiation safety expert should be consulted before the Ⅵ Special equipment may be used to contain or control autopsy to evaluate potential exposure, to recommend chemical fumes, splatters, or biological aerosols protective equipment, and to determine what procedures Emphasis is on safe practices and procedures Ⅵ Restrictions on smoking, eating, and drinking in the may be necessary to allow safe release of the body to autopsy area are enforced to reduce ingestion potential funeral home personnel. Prosectors need to prevent con- Ⅵ Gloves, gowns, and aprons are worn tamination of the cadaver cart, the route from the pa- Ⅵ Other personal protective equipment is worn as needed (e.g., to protect mucous membranes) tient’s room to the autopsy room, and the autopsy facil- Ⅵ Handwashing after removal of gloves and before leaving ity. The autopsy facility and instruments need to be the work area is required appropriately decontaminated. Radiation levels and per- Ⅵ Instruments and work surfaces are decontaminated and cleaned sonnel exposures need to be monitored and documented. Ⅵ Waste is decontaminated or processed for incineration Autopsy personnel need to be evaluated for contamina- Ⅵ Samples are labeled (including hazard warnings) and tion before they leave the autopsy area (147). contained for transport to other locations Policy issues include Ⅵ A qualified person provides supervision Electronic Devices Ⅵ Immunizations are offered (e.g., HBV); medical services are available Implantable cardioverter-defibrillators are used to Ⅵ Standard operating procedures (with biosafety issues treat tachyarrhythmias. Prosectors who manipulate these addressed) are developed devices or cut the leads can sustain an electrical dis- Facility requirements include Ⅵ The location is away from public areas; doors are lockable charge of 25 to 40 J. Their presence may not always be Ⅵ Consideration is given for directional inward airflow without suspected before the autopsy. If such a device is found, recirculation to other areas the procedure should be stopped until it can be deacti- Adapted from reference (21). vated. Manufacturers have service representatives available to assist with the deactivation process (149). dard Precautions). The increased containment of Bio- safety Level 3 (Table 3) provides protection when a risk AUTOPSY PRECAUTIONS of exposure to agents transmissible by aerosols is present (e.g., M. tuberculosis, rabies virus, and Y. pestis) (21). Risk Assessment Although the agents of the viral hemorrhagic fevers are Safety guidelines for autopsy personnel indicate that classified as Biosafety Level 4, bodies potentially in- any autopsy can potentially harbor a risk for prosectors fected with these agents can be safely autopsied using the (150). Although agent-specific degrees of risk (biosafety barrier precautions of BSL-2 combined with the negative levels) have been clearly established for biomedical and airflow and respiratory precautions of BSL-3. microbiologic laboratories, the same standards have not been well articulated for autopsy facilities. However, the Personal Protective Equipment biosafety principles that have been developed for clinical Autopsy workers need protection from bloodborne laboratories, biomedical research laboratories, and ani- and aerosol-transmissible pathogens. To protect the eyes, mal facilities (21) can be broadly applied to autopsy skin, and mucous membranes, all prosectors and autopsy activities. observers should wear a surgical scrub suit, surgical cap, Achieving appropriate worker protection from biohaz- impervious gown or apron with full sleeve coverage, ards involves personal protective equipment, engineering some form of eye protection (goggles or face shield), controls, and work practices and procedures instituted as shoe covers, and double surgical gloves (Fig. 1) (20,151– a result of risk assessment. The barriers and procedures 153). Metal and synthetic mesh gloves worn underneath used to protect health care workers from blood and body surgical gloves may mitigate the risk of autopsy injuries fluid pathogens were formerly termed Body Substance from scalpels and other sharp objects, but they offer no Isolation Procedures or Universal Precautions. Recently, protection from needle punctures (154,155). these precautions were combined into Standard Precau- Surgical masks may mitigate the risk from splashed tions, which were developed to reduce the transmission body fluids (i.e., droplets) and will help keep prosectors’ of all pathogens from moist body substances (151). Bio- hands from contacting their noses or mouths. However, safety Level 2 (BSL-2) (Table 2) provides personnel because of substantial marginal air leakage, standard protection against most bloodborne pathogens (i.e., Stan- surgical masks worn conventionally do not protect au-

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TABLE 3. Principles of Biosafety Level 3 be worn during the examination of persons who have Suitable for work with indigenous or exotic agents that died of conditions such as the viral hemorrhagic fevers can cause serious or potentially lethal disease as a and plague (59,63,83). result of exposure by the inhalation route Ⅵ Personnel receive specific training in handling materials When volatile chemicals are suspected, organic vapor (potentially) infected with pathogenic and potentially lethal cartridges must be used. These cartridges may be sand- agents wiched with a HEPA-filter cartridge for use in a PAPR Ⅵ Supervision is provided by competent scientists who are experienced in working with such agents (125). Personnel who need to wear respirators are re- Ⅵ Localized containment or ventilation devices (e.g., quired to receive medical clearance and training in downdraft tables) are used to control or contain fumes, proper fit-testing (157,158). splatters, or biologic aerosols Ⅵ Special engineering controls and appropriate personal protective clothing and equipment (including respirators) Autopsy Procedures are required and used When prosectors use safe procedures, the risks for In addition to the principles of BSL-2 Ⅵ Additional medical surveillance procedures might be autopsy-transmitted infections can be decreased. To applicable (e.g., periodic TB skin testing, serum collection avoid percutaneous injury, prosectors must be careful and testing) with sharp instruments such as scalpels, needles on sy- Ⅵ Biohazard warning signs indicating suspect agents and necessary precautions are posted Ⅵ All personnel demonstrate proficiency in the practices and procedures specific to the nature of the hazard Facility requirements include the following Ⅵ A separate room is recommended; otherwise, only persons involved with the specific autopsy are allowed in the room; room doors are lockable Ⅵ Exhaust from the autopsy room is discharged to the outside Ⅵ Ventilation to the autopsy room is balanced to provide directional airflow into the room (negatively pressured room) Ⅵ Access to a personal shower is available close to the autopsy room Ⅵ Interior surfaces of the walls, floors, and ceilings are constructed for easy cleaning and decontamination Ⅵ Floors should be monolithic and slip resistant Ⅵ Penetrations through floors, walls, and ceilings should be sealed; openings around ducts and doors are sealable to facilitate decontamination Ⅵ Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent Ⅵ Containment features of the BSL-3 autopsy room need to be verified by facility engineers and biosafety personnel before work is performed and annually thereafter Adapted from reference (21). topsy participants from inhaling airborne contaminants (156). When a risk of an aerosolized pathogen such as M. tuberculosis is present, these individuals should wear N-95 respirators (Fig. 2) at a minimum (69). The fabric of these masklike respirators is designed to filter 95% of particles that are 1 ␮m in diameter. The use of these inexpensive and comfortable respirators should be considered for all autopsies because it is frequently impossible to determine the risk for an aerosolized pathogen before an autopsy. Autopsy prosectors and observers who cannot wear N-95 respirators because of beards are advised to wear powered air-purifying respirators (PAPRs) equipped with appropriate N-95 or high-effi- ciency particulate air (HEPA) cartridge filters (Fig. 3). Both N-95 and HEPA respirators are currently available as disposable products. HEPA-filtered PAPRs can provide an additional measure of respiratory protection for FIG. 1. Standard personal protective equipment for high-risk autopsies (69). Respirators of this type should autopsy.

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FIG. 2. N-95 respirator. ringes, and suturing needles (151). Furthermore, prosectors must be aware of the potential for other sharp objects such as broken glass, splintered bone, and sharp projectiles that can be encountered during dissection (9,10). All sharp objects, including scalpels and needles, should be discarded into puncture-resistant “sharps” containers immediately after use and not be reused. Needles should not be recapped with two hands or by the use of any other method that involves pointing the needle toward any part of the handler’s body. Instead, a one-handed “scoop” technique or a mechanical device for holding the needle sheath should be used. Sharps containers should be available in the use area (151). Prosectors should thoroughly and immediately wash any skin surfaces that are contaminated with blood or other potentially infectious body fluids to prevent transmission of pathogens. Addition- ally, when an autopsy is completed, participants should wash their hands after they remove their gloves because inapparent defects may occur in gloves during use, and hands can become contaminated during glove removal (151). Using a manual saw or an oscillating saw equipped with an integrated exhauster can reduce exposure to aerosolized bone dust and pathogens (73). Instruments used in autopsy procedures should be decontaminated before cleaning, by liquid chemical soaks (typically 1:10 or 1:100 dilutions of bleach) or by autoclaving (150). Sharp instruments should be placed in

FIG. 3. Prosectors wearing powered air air-purifying respirators. (A) front view of hooded gown, (B) rear view of filter device with air supply hose extending to hood.

Am J Forensic Med Pathol, Vol. 23, No. 2, April 2002 BIOSAFETY CONSIDERATIONS FOR AUTOPSY 115 rigid containers to minimize puncture hazards to persons to bloodborne and aerosolized pathogens. Autopsy “downstream” from the autopsy room (21). Paper prod- rooms should have a minimum of 12 air exchanges per ucts, sponges, waste tissue, and similar materials can be hour and should be under negative pressure in relation to treated as standard hospital “red bag” waste and removed the surrounding spaces (69). Increasing ventilation above for terminal treatment (e.g., incineration) (150). this standard yields little additional benefit of decreased Autopsy surfaces should be decontaminated with an risk, especially if the exposure level is high (164). The air appropriate liquid chemical. If bleach is used, the con- in autopsy rooms should flow unidirectionally from centration should be adjusted: heavy blood/body fluids clean areas to less clean areas. The air should then be should be treated with undiluted bleach, moderate splat- exhausted directly to the outside of the facility (69). ter areas with 1:10 (0.5%) bleach, and usually clean areas Other equipment that can decrease exposure to aero- with 1:100 (0.05%) bleach (150,159). Agents such as M. solized pathogens includes downdraft autopsy tables, tuberculosis should be decontaminated with liquid tuber- ultraviolet irradiation devices to sterilize air, HEPA fil- culocidal solutions such as phenolic compounds tration, and biologic safety cabinets for handling infected (160,161), and prions, with NaOH (see Agent Summary tissues. Downdraft table ventilation has proved effective Statement on Prions at end of article). in reducing formaldehyde exposure during anatomy dissection (165). Pathologists can use similar downdraft Vaccines, Employee Health Surveillance, and dissection tables to examine fixed autopsy tissues or to Antibiotic Prophylaxis perform autopsies on bodies that harbor potentially aero- A vaccine to prevent hepatitis B became available in solizable pathogens. Important safety concerns are asso- 1982. Vaccination is currently recommended for all ciated with using ultraviolet irradiation (69). Biologic health care workers who are regularly exposed to blood safety cabinets can be used to examine tissues potentially and other body fluids and can significantly decrease the contaminated by infectious agents (21,166). When com- risk of occupational exposure to this pathogen (20,31). bined with appropriate techniques, Class I and Class II The U.S. Department of Labor and the U.S. Department biologic safety cabinets can effectively contain moderate of Health and Human Services require employers to and high-risk microorganisms (Biosafety Level 2 and 3 provide this vaccine at no charge to at-risk health care agents). These cabinets have inward directed airflow workers (20). Other vaccines are available for diseases velocities of 75 to 100 linear feet per minute to protect potentially transmitted at autopsy (e.g., plague and ra- workers from infectious aerosols generated within the bies). However, none of these vaccines are presently cabinet. Class III cabinets which contain hazardous ma- recommended for unexposed autopsy workers with a low terials in a totally enclosed, ventilated space protected by risk of encountering these infections. HEPA filters, are most appropriate for work with haz- Autopsy workers should have a baseline tuberculin ardous Biosafety Level 4 agents. If biosafety cabinets are skin test at the time of employment. Periodic retesting of ducted to the outside exhaust system, they may be used workers with negative skin test results should occur at to examine specimens contaminated by toxic or volatile regular intervals based on an assessment of risk. In chemicals or radionuclides (21). Chemical fume hoods addition, there should be retesting whenever there is can also be effective primary containment devices for exposure to a tuberculous patient without appropriate tissues contaminated by toxic or volatile chemicals precautions. A history of a positive skin test result or of (114). These cabinets and fume hoods need to be certi- tuberculosis should exempt the worker from further test- fied in situ at the time of installation, any time the device ing (69). is moved, and at least annually thereafter. Personnel Under certain conditions, administering prophylactic must also be trained in their use (21). antibiotics to autopsy workers exposed to potentially lethal infectious organisms may be appropriate. For ex- PRESENT STATUS OF AUTOPSY FACILITIES ample, persons exposed to Y. pestis aerosols should re- IN THE UNITED STATES ceive a course of antimicrobial therapy, regardless of vaccine status (162). Most medical examiner, coroner, and hospital autopsy facilities in the United States are not constructed to Facility Design enhance autopsy biosafety (167). Many autopsy rooms Autopsy facility design is complicated and autopsy are located in aging facilities, often with shared ventila- facility construction is expensive. However, from the tion between prosecting and administrative space. Most biosafety standpoint, autopsy rooms should have a sep- autopsy rooms barely meet the design criteria of Bio- arate air supply and should be physically separated from safety Level 2. Only a few are constructed to meet the administrative areas of the facility (163). Separation Biosafety Level 3 requirements. At present, the U.S. prevents employees and other persons who do not par- Army Medical Research Institute of Infectious Diseases ticipate in postmortem examinations from being exposed in Fort Detrick, Maryland, has the only autopsy facility

Am J Forensic Med Pathol, Vol. 23, No. 2, April 2002 116 K. NOLTE ET AL. in the United States that can function at Biosafety Level health care after exposure to bloodborne and aerosolized 4. Although this facility was designed for human autop- pathogens or toxic chemicals. sies, it is currently used for nonhuman primate examinations (NK Jaax, personal communication, 1999). Further- AGENT SUMMARY STATEMENT ON PRIONS more, many autopsy pathologists have been slow in complying with even basic precautions promulgated to Prions are proteinaceous infectious particles that lack prevent the transmission of both bloodborne and aero- nucleic acids (171). Prions are composed largely, if not solized pathogens (20,69,168,169). entirely, of an abnormal isoform of a normal cellular protein. In mammals, prions are composed of an abnormal, pathogenic isoform of the prion protein (PrP), des- RECOMMENDATIONS ignated PrPSc. The “Sc” superscript was initially derived We need to have a better prepared national autopsy from the term scrapie because scrapie is the prototypic infrastructure that is uniformly capable of investigating prion disease. Because all of the known prion diseases of fatalities resulting from contagious and toxic agents. mammals involve aberrant metabolism of PrP similar to Achieving this goal will require substantial changes in that observed in scrapie, use of the “Sc” superscript is autopsy facilities, in policies and procedures, and in the suggested for all abnormal pathogenic PrP isoforms protective equipment provided to prosectors. Funding (172). In this context, the “Sc” superscript is used to should be made available to effect these changes and to designate the scrapie-like isoform of PrP. bring autopsy facilities into compliance with accepted A chromosomal gene encodes PrP, and no PrP genes public health standards (20,21,69). All autopsy facilities are found in purified preparations of prions. PrPSc is should be able to function at Biosafety Level 3. In derived from PrPC (the cellular isoform of PrP) by a facilities lacking proper ventilation, personal protective posttranslational process whereby PrPSc acquires a high equipment such as N-95 respirators, coupled with the beta-sheet content (173). Neither prion-specific nucleic practices and procedures of BSL-3, can provide adequate acids nor virus-like particles have been detected in pu- worker protection against aerosolized pathogens. How- rified, infectious preparations. In fungi, evidence for ever, providing this equipment does not address the issue three different prions has been accumulated (174). The of how to protect nonautopsy staff working elsewhere in mammalian prions cause scrapie and other related neu- these buildings. When the autopsy room is not negatively rodegenerative diseases in humans and animals. The pressured and the air is not properly exhausted outside prion diseases are also referred to as the transmissible (i.e., the principal secondary containment features of spongiform encephalopathies (175). Biosafety Level 3), protecting nonautopsy staff from aerosolized pathogens is impossible. Consequently, Bio- Autopsies safety Level 3 should be considered the standard for Routine autopsies should be performed using BSL-2 every autopsy facility. precautions (176) augmented by BSL-3 facility ventila- To achieve this goal, new autopsy facilities should be tion and respiratory precautions. At autopsy, the entire constructed with adequate ventilation to ensure protec- brain should be collected and cut into coronal sections tion of prosectors and other building occupants. Substan- about 1.5 inches (ϳ4 cm) thick; small blocks of tissue dard facilities need to be remodeled to reach an effective can easily be removed from each coronal section and level of protection. Because new construction and facil- placed in fixative for subsequent histopathologic analy- ity remodeling are expensive, and hospital autopsy rates ses. Each coronal section is immediately heat-sealed in a are declining nationally, some thought should be given to heavy-duty plastic bag. The outside of this bag is as- regionalizing autopsy services. A mobile containment sumed to be contaminated with prions and other patho- autopsy facility constructed to operate at Biosafety Lev- gens. With fresh gloves or with the help of an assistant els 3 or 4 may be useful in providing autopsy support to with uncontaminated gloves, the bag containing the spec- jurisdictions with inadequate facilities when they are imen is placed into another plastic bag that does not have confronted with contagious or toxic cases. Plastic film a contaminated outer surface. The samples should then tent isolators with sleeved glove ports and negative pres- be frozen on dry ice or placed directly in a Ϫ70°C sure ventilation have been used for Biosafety Level 3 freezer for storage. At the very minimum, a coronal autopsies (170). Perhaps such mobile facilities or devices section of cerebral hemisphere containing the thalamus should be a resource offered by the federal government. and one of the cerebellar hemisphere and brainstem In addition, all agencies that perform autopsies should should be taken and frozen. provide prosectors with adequate respirators and other The absence of any known effective treatment for personal protective devices, appropriate biosafety train- prion disease demands caution. The highest concentra- ing, immunization against hepatitis B virus, periodic tions of prions are in the central nervous system and its screening for tuberculosis, and access to appropriate coverings. On the basis of animal studies, high concen-

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TABLE 4. Precautions for autopsies of subjects with TABLE 6. Brain cutting procedures suspected prion disease 1. After adequate formaldehyde fixation (at least 10–14 days), 1. Attendance should be limited to at least one experienced the brain is examined and cut on a table covered with an pathologist and minimal staff. One of the staff avoids direct absorbent pad with an impermeable backing. contact with the deceased but assists with the handling of 2. Samples for histology are placed in cassettes labeled “CJD instruments and specimen containers. precautions.” For laboratories that do not have embedding 2. Standard autopsy attire is mandatory. and staining equipment or microtome dedicated to a. A disposable, waterproof gown is worn in place of a infectious diseases, including Creutzfeldt-Jakob disease, cloth gown. blocks of formalin-fixed tissue can be placed in 96% b. Cut-resistant gloves are worn underneath two pairs of absolute formic acid for 30 minutes, followed by fresh 10% surgical gloves, or chain mail gloves are worn between neutral buffered formalin solution for at least 48 hours. The two pairs of surgical gloves. tissue block is then embedded in paraffin as usual. c. Aerosols are mainly created during opening of the skull Standard neurohistologic or immunohistochemical with a Stryker saw. Appropriate respiratory protection techniques are not obviously affected by formic acid should be worn (i.e., powered air-purifying respirators). treatment; however, tissue sections are brittle and crack 3. To reduce contamination of the autopsy suite during sectioning. a. The autopsy table is covered with an absorbent sheet 3. All instruments and surfaces that come into contact with that has a waterproof backing. the tissue are decontaminated as described in Table 5. b. Contaminated instruments are placed on an absorbent 4. Tissue remnants, cutting debris, and contaminated pad. formaldehyde solution should be discarded within a plastic c. The brain is removed while the head is in a plastic bag container as infectious hospital waste for eventual to reduce aerosolization and splatter. incineration. d. The brain can be placed into a container with a plastic bag liner for weighing. e. The brain is placed onto a cutting board, and appropriate samples are dissected for snap freezing. punctures, exposure to aerosols, and contamination of f. The brain or organs to be fixed are immediately placed the autopsy room, morgue surfaces, and instruments. into a container with 10% neutral buffered formalin. g. In most cases of suspected prion disease, the autopsy Unfixed samples of brain, spinal cord, and other tissues can be limited to examination of the brain only. In cases containing human prions should be processed with ex- requiring a full autopsy, consideration should be given to treme care in a laboratory operating at Biosafety Level 3. examining and sampling thoracic and abdominal organs in situ. The processing of small amounts of formalin-fixed tissues containing human prions require Biosafety Level 2 precautions (176). trations of prions are also likely found in spleen, thymus, lymph nodes, and lung. The main precaution to be taken TABLE 7. when working with prion-infected or contaminated ma- Tissue preparation terial is to avoid puncture of the skin (177). Although 1. Histology technicians wear gloves, apron, laboratory coat, and face protection. there is no documentation of the transmission of prions 2. Adequate fixation of small tissue samples (e.g., biopsy to humans through droplets of blood or cerebrospinal specimens) from a patient with suspected prion disease is fluid, or by exposure to intact skin, or gastric and mucous followed by postfixation in 96% absolute formic acid for 30 minutes, followed by 48 hours in fresh 10% formalin. membranes, the risk of such occurrences is a possibility. 3. Liquid waste is collected in a 4-L waste bottle containing The prosector should wear cut-resistant gloves whenever 600 ml 6N sodium hydroxide. possible. If accidental contamination of the skin occurs, 4. Gloves, embedding molds, and all handling materials are disposed of as biohazardous waste. swab the area with 1 N sodium hydroxide for 5 minutes 5. Tissue cassettes are processed manually to prevent and then wash with copious amounts of water. Tables 4 contamination of tissue processors. through 7 provide guidelines for reducing the risk of skin 6. Tissues are embedded in a disposable embedding mold. If used, forceps are decontaminated. 7. In preparing sections, gloves are worn; section waste is collected and disposed of in a biohazard waste receptacle. TABLE 5. Autopsy suite decontamination procedures The knife is wiped with 1–2N NaOH, and the knife used is 1. Instruments (open box locks and jaws) and saw blades are discarded immediately in a “biohazard sharps” receptacle. placed into a large stainless steel dish, soaked for 1 hour in Slides are labeled “CJD precautions.” The sectioned block 2N sodium hydroxide or 2 hours in 1N sodium hydroxide, is sealed with paraffin. and then rinsed thoroughly in water before autoclaving at 8. Routine staining: 134°C (gravity displacement steam autoclaving for 1 hour; a. Slides are processed by hand. porous load steam autoclaving for one 18-minute cycle at b. Reagents are prepared in 100-ml disposable specimen 30 lb psi or six 3-minute cycles at 30 lb psi). cups. 2. The Stryker saw is cleaned by repeated wetting with 2N c. After placing the coverslip on, slides are decontaminated sodium hydroxide solution over a 1-hour period. by soaking them for 1 hour in 2N NaOH. Appropriate washing to remove residual NaOH is required. d. Slides are labeled “Infectious—CJD.” 3. The absorbent table cover and instrument pads, disposable 9. Other suggestions: clothing, etc., are double bagged in appropriate infectious a. Disposable specimen cups or slide mailers may be used waste bags for incineration. for reagents. 4. Any suspected areas of contamination of the autopsy table b. Slides for immunocytochemistry may be processed in or room are decontaminated by repeated wetting over 1 disposable Petri dishes. hour with 2N sodium hydroxide. c. Equipment is decontaminated as described above.

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Bovine Spongiform Encephalopathy Although no evidence suggests that aerosol transmis- The risk of infection for humans by bovine spongi- sion occurs in the natural disease, it is prudent to avoid form encephalopathy prions is unclear. Perhaps the most the generation of aerosols or droplets during the manip- prudent approach is to study these prions in a Biosafety ulation of tissues or fluids and during the necropsy of Level 2 or 3 facility (depending on the samples to be experimental animals or infected humans. It is further studied), as noted above for human prions (i.e., Biosafety strongly recommended that gloves be worn for activities Level 3 for brain, spinal cord, and other organs with high that provide the opportunity for skin contact with infec- prion concentrations.) tious tissues and fluids. Formaldehyde-fixed and paraffin- embedded tissues, especially of the brain, remain infectious. Some investigators recommend that formalin-fixed Physical Properties of Prions tissues from suspected cases of prion disease be im- The smallest infectious prion particle is probably a mersed for 30 minutes in 96% formic acid or phenol be- dimer of PrPSc; this estimate is consistent with an ioniz- fore histopathologic processing (186), but such treatment ing radiation target size of 55 Ϯ 9 kDa (178). Therefore, may severely distort the microscopic neuropathology. prions may not be retained by most of the filters that efficiently eliminate bacteria and viruses. Additionally, prions aggregate into particles of nonuniform size and Handling and Processing of Tissues from Patients cannot be solubilized by detergents, except under dena- with Suspected Prion Disease turing conditions where infectivity is lost (179,180). Pri- The special characteristics of work with prions require ons resist inactivation by nucleases (181), ultraviolet particular attention to the facilities, equipment, policies, irradiation at 254 nm (182,183), and treatment with pso- and procedures involved. Handling and processing tis- ralens (184), divalent cations, metal ion chelators, acids sues from patients with suspected prion disease must be (between pH 3 and 7), hydroxylamine, formalin, boiling, done with precautions equal to those used with prion- or proteases (185,186). infected experimental animals. The related considerations outlined in Tables 4 through 7 should be incorpo- rated into the laboratory’s risk management protocols for Inactivation of Prions this work. Prions are characterized by extreme resistance to con- ventional inactivation procedures, including irradiation, Acknowledgments: Jiri Safar, M.D., Darlene Groth, A.B., boiling, dry heat, and chemicals (formalin, betapropio- Stephen J. DeArmond, M.D., Ph.D., and Stanley B. Prusiner, lactone, and alcohols). Whereas prion infectivity in pu- M.D., of the University of California San Francisco, San Fran- rified samples is diminished by prolonged digestion with cisco, California, prepared the Agent Summary Statement on proteases (187,188), the results from boiling in sodium Prions, which the authors adapted from a noncopyrighted source (21). dodecyl sulfate and urea are variable. Sterilization of All rights reserved. Adapted and reprinted with permission rodent brain extracts with high titers of prions requires from the American Biological Safety Association (ABSA), autoclaving at 132°C for 4.5 hours. Denaturing organic Mundelein, IL. Originally published in Anthology of Biosafety solvents such as phenol, or chaotropic reagents such as IV: Issues in Public Health (pp. 1–50). Copyright © 2001. guanidine isothiocyanate, or alkali such as NaOH can also be used for sterilization (189–193). 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